Epilepsy affects 1 in 26 Americans over the course of a lifetime. Temporal lobe epilepsy is one of the most common types. Medical treatment fails in one-third of patients. Development of effective therapies is limited by gaps in knowledge about how spontaneous seizures begin. Determining how spontaneous seizures initiate in temporal lobe epilepsy is our long-term goal. The primary goal of the proposed project is to test the hypothesis that seizures start, at least in part, because inhibitory interneurons reduce their action potential firing frequency (deactivate), which reduces inhibitory control of excitatory neurons. Aim 1 is to test whether interneuron deactivation is seizure specific. Local field potential recordings from many brain regions in epileptic pilocarpine- treated rats would be used to identify sites of seizure initiation. Simultaneous unit recordings of hippocampal interneurons would be evaluated to test whether interneuron deactivation is more frequent and severe in epileptic versus control rats and at seizure onset sites versus non-onset sites. Aim 2 is to test whether preventing interneuron deactivation prevents seizures. Local field potential recordings from many brain regions would be used to identify rats in which the ventral hippocampus is the seizure focus. Closed-loop activation of interneurons would prevent deactivation at sites of seizure onset. Optogenetics and Cre-dependent channelrhodopsin-expressing viral vectors in glutamic acid decarboxylase-Cre and parvalbumin-Cre transgenic rats would be used to selectively activate specific interneuron subtypes. Optogenetic stimulation would be triggered by real-time detection of rapid eye movement sleep, which precedes many seizures in rat models of temporal lobe epilepsy. Aim 3 is to test whether deactivating interneurons causes seizures. The experimental approach would be similar to that of Aim 2, except the novel Cl--conducting opsin SwiChR++ would be used to deactivate interneurons. Our deactivation hypothesis is opposite of others that contend that hyperactivity of interneurons is a mechanism of seizure initiation. The proposed project would rigorously test multiple hypotheses of temporal lobe ictogenesis and advance understanding of how spontaneous seizures start.